HIV-1 replication in gut-associated lymphoid tissue (GALT) in the early stages of infection results in massive depletion of mucosal effector CD4+ T cells, in particular IL-17-producing CD4+ T cells (Th17). Depletion of Th17 cells leads to bacterial translocation from the lumen into the lamina propria (LP) and subsequently the circulation, a process associated with increased immune activation and inflammation. Thus, understanding how HIV-1 induces GALT CD4+ T cell death may be key to preventing the inflammatory sequelae of HIV- 1 infection. However, the mechanism by which HIV-1 kills CD4+ T cells remains one of the most critical unanswered questions in basic HIV/AIDS research. Despite extensive published studies on the subject, most prior CD4+ T cell killing studies utilized X4-tropic HIV-1 strains and cell types that are not directly relevant to understanding CD4+ T cell depletion in mucosal compartments. To address this issue, we recently developed a novel experimental system to model the interactions between R5-tropic HIV-1, microbial species and LP CD4+ Th cell subsets ex vivo using primary intestinal mucosal cells. Thus, our collaborative team is in a unique position to address the following critical knowledge gaps in HIV-1 mucosal immunopathogenesis: 1. How does R5-tropic HIV-1 cause GALT CD4+ T cell death? 2. How do translocating enteric bacteria influence HIV-mediated GALT CD4+ T cell killing? 3. Which viral determinants mediate GALT CD4+ T death by transmitted/founder HIV-1 strains? Using our dynamic ex vivo LP infection model, we propose three specific aims to address these important questions. In Aim 1, we will utilize differentially-labeled HIV-1 infected and uninfected cells to investigate the role of PCD pathways in HIV-1 mediated CD4+ T cell death by counteracting caspase activity, inhibiting HIV-1 at discrete steps in its life cycle, and using non-biased gene expression profiling. In Aim 2, we will determine the biological impact of representative gut bacterial species individually, and in combination, on HIV-1 mediated LP CD4+ T cell death and identify the immunomodulatory properties of bacteria that influence CD4+ T cell depletion. In Aim 3, we will document the replication and killing characteristics of a panel of 40 Transmitted/Founder and chronic HIV-1 Subtype B and C infectious molecular clones, to define the viral characteristics responsible for LP CD4+ T cell death. The results of these studies may ultimately provide insight into pathways that can be targeted to preserve or restore the integrity of the mucosal immune system in HIV- infected patients and thereby limit systemic inflammation and its downstream consequences.